Adrenergic mechanism in the control of endothelial function.

There is considerable evidence that many disease are associated with endothelial dysfunction and reduced nitric oxide production such as hypertension, obesity, dyslipidemias, diabetes, heart failure, atherosclerosis. Notably these conditions are also characterized by alteration in the adrenergic tone. Whether these two mechanisms are just epiphenomenal each other or there is a functional link, it is still to be established. A starting ground to establish this issue is that vascular endothelium plays an important role in the function of cardiovascular system and that adrenergic receptors on endothelial cells contribute to the regulation of vasomotor tone. The aim of this excerpt is to review current knowledge on the physiology of endothelial adrenergic receptors to contribute to the basis for newer and better approaches to endothelial dysfunction in the setup of cardiovascular conditions.


Introduction
The endothelium controls several vascular functions, including vasculature tone and permeability, thrombosis, hemostasis and angiogenesis [1][2][3][4] . It is noteworthy that all these functions can be regulated by the activation of receptors and often the same receptor can activate multiple endothelial functions. The adrenergic system is the major regulator of cardiac and vascular function and of endothelial vasorelaxation by means of  and  adrenergic receptors activation. The adrenergic receptors (ARs) are part of a large family of G protein coupled receptors (GPCR) which mediate the functional effects of catecholamines like epinephrine and norepinephrine. The ARs family includes three  ( 1 ,  2 ,  3 ), three  1 ( 1A ,  1B ,  1D ) and three  2 ( 2A ,  2B ,  2C ) receptor subtypes. These receptors actively participate to the release of nitric oxide (NO) in order to regulate endothelial function 5 . NO plays a crucial role in endothelium homeostasis, with important vasodilatory, anti-thrombotic and anti-atherogenic properties. NO mediates most of the endothelial functions: it has been invoked as a mechanism in vasorelaxation, endothelium permeability and neoangiogenesis 3 . NO in the endothelium is constitutively produced by the endothelial NO synthase, eNOS 6 . This latter is then further activated through calcium levels 7 and phosphorylation of various serine residues by a number of protein kinases 8,9 . Indeed, it has been demonstrated that NO is activated by means of the PI3K pathway in response to the stimulation of tyrosine kinase 10,11 .
The impaired ability of vascular endothelium to stimulate vasodilation is referred to as "Endothelial Dysfunction" and the major cause is the decreased bioavailability of NO in different conditions which can be due to various mechanisms: reduced eNOS expression, altered NO production and increased NO catabolism. Endothelial dysfunction plays a key role in the development of cardiovascular disease such as hypertension, type 2 diabetes and heart failure. The identification of the underlying pathogenic mechanisms will lead to the discovery of newer and more potent tools to Università degli Studi di Salerno treat such diseases. On this issue, endothelial dysfunction has been associated to signal transduction abnormalities observed in hypertension. In particular, adrenergic vasorelaxation has been demonstrated to be impaired in hypertensive patients, probably due to the presence of increased desensitization and impaired signalling of AR. Adrenergic receptors on endothelium have been longely not considered functional to the regulation of the vascular tone. On the contrary, it is possible to identify very specific roles for such receptors in several endothelial function. This review will summarize the effects of adrenergic receptors on endothelial functions, focusing on modulation of NO synthesis and angiogenesis.

 adrenergic receptors
AR are GPCRs that couple to Gq protein. The Gq subunit is a primary activator of phospholipase C (PLC). Activation of PLC promotes the cleavage of the inositol substrate phosphatidyl-inositol 4,5 bisphosphate (PIP2) to yield diacylglycerol (DAG) and inositol 1,4,5trisphosphate (IP3). DAG and IP3 promote the activation of a protein kinase C (PKC).  1 AR can also activate specific adenylate (adenylyl) cyclases (AC) leading to an increase in cAMP levels. The activation of specific PLCs and ACs requires a complex balance of signals from G-proteins, especially the G subunits, within specific cell contexts. DAG and cAMP are second messengers that affect a wide array of cell signaling pathways and responses.

 1 AR and Nitric oxide
Several reports 12,13 have produced evidence for the functional presence of vasorelaxant  1 AR in the brachial and pulmonary arteries isolated from the rabbit and rat, respectively. According to these reports, the pharmacological stimulation of  1 AR located on endothelial cells, is able to generate NO, whereas the stimulation of  2 AR releases a relaxing prostanoid 12,13 . Filippi demonstrated that nanomolar concentrations of phenylephrine, which are devoid of any contractile effect, induced a Università degli Studi di Salerno slight endothelium-dependent vasorelaxation in the rat mesenteric vascular bed through the stimulation of  1D AR, located on endothelial cells, which act through phospholipase C stimulation, followed by IP1 generation, and nitric-oxide synthase activation. Conversely, the increase in perfusion pressure induced by micromolar concentrations of phenylephrine is attributable to the stimulation of  1A AR 14 .

 1 AR and angiogenesis
Neo-angiogenesis has long been known to be a highly ordered multistep molecular process under tight regulation by endothelial cells 15 and closely associated with endothelial cell proliferation and migration and to the capability of these cells to modulate the levels of VEGF, the most important cytokine system involved in the formation of new vessels 16 . A series of biological, chemical, hormonal effectors can interfere with this process. Several data support the notion that α 1adrenergic receptor should also be ranked among these agents. Indeed, it has been demonstrated that the  1A -and the  1B -AR subtypes but not the  1D subtype are expressed in cultured rat aorta endothelial cells. The activation of these  1 -AR in endothelial cells provide a negative regulation of angiogenesis 17 . Indeed, pharmacological antagonism of  1 -AR in endothelial cells from WKY rats by doxazosin enhanced, while stimulation of these adrenergic receptors with phenylephrine, inhibited endothelial mechanisms of angiogenesis such as cell proliferation and DNA synthesis, ERK and retinoblastoma protein (Rb) phosphorylation, cell migration and tubule formation 17 . A similar phenotype can be observed in vivo, since an increased  1 -adrenergic receptor density in the ischaemic hindlimb, compared to non-ischaemic hindlimb, suggested an enhanced  1 -adrenergic receptor tone in the ischaemic tissue. Treatment with doxazosin did not alter systemic blood pressure but enhanced neo-angiogenesis in the ischaemic hindlimb 17 .

 2 ARand Nitric oxide
It has been demonstrated that  2 adrenergic agonists cause endothelium dependent relaxation, that is reduced or abolished by inhibitors of L-arginine/NO pathway. It depends on the activation of  2 AR on endothelial cells which stimulates the release of NO, an action that would tend to attenuate vasoconstriction produced by the activation of post-junctional vascular  1 AR [18][19][20] . The  2 AR subtype that cause endothelium dependent relaxation belongs to the  2A/D subtype, despite the 21 . It appears that this ratio may not be constant, since it varies within the vascular bed. Indeed, Bockman demonstrated that in the rat mesenteric artery the  2 AR is coupled to endothelium dependent NO-mediated relaxations and belongs to the  2A/D subtype appearing in its  2D version 22 . It has been demonstrated that endothelium dependent relaxation to  2 adrenergic agonists is prevented by pertussis toxin 23-28 , suggesting the involvement of G i proteins in the signal transduction from the receptor to the activation of nitric oxide synthase 29, 30 . Indeed,  2 adrenergic agonists cause activation of G i proteins in endothelial cells and stimulate NO synthase activity 31,32 . Contrary to what expected, cAMP is not involved in the signal transduction pathway for  2A/D AR mediated NO formation 22 .
Indeed, the use of forskolin to oppose  2 adrenergic receptor mediated inhibition of cAMP formation in endothelium did not affect the relaxant response to  2 AR agonists, suggesting that cAMP is not involved in the coupling of  2 AR to NO. There are physiological modulation of endothelium dependent relaxation to  2 adrenergic agonists. Such relaxation is upregulated by chronic increase in blood flow 33 or exercise training 34 . Insulin enhances NO mediated vasorelaxation both in animal 25 and human 32 vasculature.

-adrenergic receptors
ARs signal by coupling to the stimulatory G protein, Gs, leads to the activation of adenylyl cyclase and accumulation of the second messenger cAMP 35,36 . However, recent studies indicate that under certain conditions AR, and particularly   AR, can couple to Gi as well as to Gs [37][38][39][40][41] . It is now widely accepted that AR exist on endothelial cells 10,38,40,42  . This appears to hold true through experimental models (rat or man) and vascular districts (see Figure 1).

 1 and  2 adrenergic receptors
It is now recognized that βAR located in the endothelium play an important role in the relaxant response to ISO, since the non selective β 1 -and β 2 -adrenergic receptor antagonist propranolol antagonized this relaxant effect 43,44 . However, recent studies carried out in humans, in umbilical veins in vitro 10 or in the forearm in vivo 45 , showed that vasorelaxation to ISO is abolished by the selective β 2 AR antagonist ICI-118551 and remains unchanged in the presence of the β 1 AR antagonist CGP-20712, indicating that, as in the vascular smooth muscle cells 46 , the endothelial βAR are totally or at least predominantly of the β 2 subtype 10, 45 . β 2 AR are seven transmembrane receptors coupled through G s proteins to a cAMP dependent intracellular pathway 47 . It has been demonstrated that PKA posphorylation of the third intracellular loop of the β 2 AR increases the affinity of the receptor for G i protein 48,49 . This switch leads to two consequences: first, it decreases the rate of cAMP generation, since G i activation inhibits adenylyl cyclase activity. Second, it increases non cAMP dependent signaling through G i , such as activation of the extracellular signal-regulated kinases ERK1/2 and PI 3 K 50-54 . Gi coupled receptors have been shown to regulate non-receptor tyrosine kinases, such as SRC, which acts as an intermediate between G i and other molecules like RAS and PI 3 K 53, 55 .

 2 AR and Nitric oxide
For years it has been given for granted that vascular β 2 AR mediate adrenergic vasorelaxation through direct activation of vascular smooth muscle cells 56 . However, recent data challenge this vision, and show that β 2 AR-dependent vasorelaxation is mediated at least in part, by endothelium through nitric oxide (NO) dependent processes 10 . We have recently demonstrated that the β 2 AR are expressed on endothelial cells (EC) and their stimulation causes endothelial nitric oxide synthase (eNOS) activation 57 . In particular,   AR couple to eNOS and induce NO dependent vasodilation 57 .
The mechanism of eNOS activation following β 2 AR stimulation is known to be AKT dependent 58 .

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Indeed, the activity of eNOS is regulated by both a calcium/calmodulin dependent fashion 59 and AKT dependent eNOS phosphorylation in Ser 1177 8,[60][61][62][63] . AKT is primarily activated in response to stimulation of transmembrane receptors with intrinsic tyrosine kinase activity or indirectly coupled to tyrosine kinases or to seven transmembrane G protein-coupled receptor 11,61,64 . Therefore AKT acts as integrator of different signal transduction pathways converging on eNOS, including endothelial β 2 AR receptor 9, 58, 62, 63, 65 .

 2 AR and angiogenesis
In the endothelium ARs control other important endothelial functions like angiogenesis, that is tightly associated to endothelial cell migration and proliferation 57,65,66 . We demonstrated that   AR stimulation with ISO and the overexpression of   AR increases endothelial cell proliferation.
Moreover,   AR stimulation induces ERK phosphorylation and the MEKK inhibitor, U0126, inhibits   AR induced cell proliferation 66 suggesting that   AR dependent cell proliferation is dependent on ERK activation. We studied post-ischaemic angiogenesis in the hindlimb (HL) of β 2 AR knock-out mice (β 2 AR-/-) in vivo and explored possible molecular mechanisms in vitro.
Angiogenesis was severely impaired in β 2 AR-/-mice subjected to femoral artery resection, but was restored by gene therapy with ADβ 2 AR. The proangiogenic responses to a variety of stimuli were impaired in β 2 AR-/-EC in vitro 17 . Moreover, removal of β 2 ARs impaired the activation of NFκB, a transcription factor that promotes angiogenesis; ISO did not induce NFκB activation in β 2 AR(-/-) EC 17 . ADβ 2 AR administration restored β 2 AR membrane density and reinstated the NFκB response to ISO 17 . These results suggest that β 2 ARs control angiogenesis through the tight regulation of nuclear transcriptional activity.

5.
 1 ARand  2 AR differently regulate neo-angiogenesis α 1 -and β 2 -adrenergic receptors mediate opposite effects on neo-angiogenesis, comparable to their regulation of the vascular tone. In particular, the α 1 -AR is inhibitory, whereas the β 2 -AR is stimulant to neo-angiogenesis. Interestingly, in ischaemia, the α 1 -AR are upregulated, thus causing a predominance of α 1 -adrenergic receptor signalling over that of β 2 -AR, which is downregulated.
Furthermore, in conditions such as hypertension, where the α 1 -AR tone is higher than that of the β 2 -AR, there is also an impairment in neo-angiogenesis 66,67 . It is interesting to note that in the ischaemic hindlimb, α 1 -AR blockade resulted in a normalization of β 2 -AR density together with improved neo-angiogenesis. α 1 -AR upregulation, in particular, might be a regulatory mechanism aimed at preventing excessive angiogenesis. This upregulation might be triggered by ischaemia, through regulatory sequences within the gene promoter, which have been demonstrated for both the α 1A -and α 1B -adrenergic receptor 68, 69 .

 3 adrenergic receptors
In rat thoracic aorta, Trochu showed that  3 AR are mainly located on endothelial cells and act in conjunction with  1 AR and  2 AR to mediate relaxation through activation of NO synthase pathway and subsequent increase in tissue cyclic GMP content and is reduced by endothelium removal or in presence of L-NMMA 70 . This  3 AR mediated aorta relaxation seems to be independent of G i proteins stimulation, since the blockage of G i protein by PTX does not modify  3 AR agonists induced relaxation. On the contrary, selective potassium channels blockers of K (Ca), K (ATP) and K (v) decreased  3 AR agonists induced relaxation. So it appears that this effect results from the activation of several potassium channels, K (Ca), K (ATP) and K (v) 71 .

Pathological implications
It was reported that noradrenaline-induced release of nitric oxide is enhanced in mineralcorticoid hypertension 72 indicating that  2 AR may play an important role in the regulation of vascular tone not only in physiological but also in pathological conditions. The implications of impaired βAR signalling in the pathophysiology of several cardiovascular disorders has been studied in animals and humans. Data from these studies indicate that changes in βAR function are induced by heart failure 73,74 and hypertension 75,76 . Moreover, alteration in βAR function were found also with physiological aging 77, 78 , due to receptor downregulation and desensitization. Exercise restored the impaired signalling and AR dependent vasorelaxation 79 . We and others have observed that impaired βAR signalling may account for dysfunctional βAR vasorelaxation in hypertension. In this condition, β 2 AR overexpression in hypertensive rat carotids corrects impaired vasorelaxation to AR stimulation to levels similar to those seen in normotensive rats 57 . We proved that impaired endothelium dependent vasorelaxation in spontaneously hypertensive rats (SHR) can be corrected by increasing the signal transduction pathways leading to nitric oxide synthase activation 80